The zebrafish (Danio rerio) is the preeminent non-mammalian vertebrate system for the study of core vertebrate biology and behavior and for the modeling of human disease. Collections of molecularly characterized mutant lines have been powerful enabling tools for the nematode, fly and mouse fields. Genomic approaches have robustly characterized the nuclear genome and transcriptome. However, the genomic assessment of the full complexity of the proteome in a dynamic context and in vivo is still largely unknown. The zebrafish offers the first comprehensive analysis of the proteome using as template an entire vertebrate genome. We show that mutagenic protein trap gene-breaking transposons (GBTs) are effective and revertible loss- of-function tools for exploring traditional areas such as the biology of development (including organogenesis) but also open up new options including the genetics of behavior such as the biology of the nicotine response. A panel of Cre recombinase regulatable mutant zebrafish alleles offers the opportunity to explore critical questions in vertebrate biology at the tissue, organ and cellular level of resolution. This application is in response to PAR-08-139 Enhancing Zebrafish Research with Research Tools and Techniques (R01) to complete progress on the development of a large-scale collection of these revertible mutant alleles for the zebrafish community through a coordinated international effort. We will deliver the following at project's end: I. 1000 new mutagenic protein trap GBT zebrafish lines with basic description of the tagged expression patterns will be established and made available in real-time through the searchable online database zfishbook.org. These lines are immediately available through distribution by the Mayo Clinic Zebrafish Core Facility. II. We will conduct molecular analyses of these lines and subsequent annotation on the zebrafish genome project to identify the trapped proteins in this collection. We will continue to use our successful next generation sequence techniques followed by linkage analysis to isolate and map GBT insertions on the zebrafish genome. All sequence information will be initially distributed through zfishbook.org and then integrated in ZFIN, Fishmap and other zebrafish genomic databases. III. We will regularly synchronize this collection with the Zebrafish International Resource Center (ZIRC) for long-term archival and distribution to the zebrafish community. We will use a recently established 2D barcoding inventory system with a state-of-the-art sperm cryopreservation approach to facilitate regular shipping of new lines to ZIRC. This proposal is to complete this molecularly characterized collection, in silico catalog and distribution network of revertible mutations for the preeminent non-mammalian vertebrate, the zebrafish. These dominantly marked mutant lines also represent an ideal substrate for large-scale behavioral and other phenotypic assessment of the genome as a part of the broader initiative to functionally annotate the vertebrate genome using the zebrafish.